The forkhead transcription factor FOXO1 stimulates the expression of the adipocyte resistin gene

General and Comparative Endocrinology 196 (2014) 41–51

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The forkhead transcription factor FOXO1 stimulates the expression of the adipocyte resistin gene q Chi-Wei Liu a,1, Shu-Ya Yang a,1, Cheng-Kuo Lin b,2, Hang-Seng Liu b, Low-Tone Ho c, Liang-Yi Wu d, Meng-Jung Lee a, Hui-Chen Ku a, Hsin-Huei Chang a, Rong-Nan Huang e, Yung-Hsi Kao a,⇑ a

Department of Life Sciences, National Central University, Jhongli, Taoyuan, Taiwan Department of Joint Laboratory, Armed Forces Taoyuan General Hospital, Taoyuan, Taiwan Department of Internal Medicine, Veterans General Hospital, Taipei, Taiwan d Department of Bioscience Technology, Chung-Yuan Christian University, Jhongli, Taoyuan, Taiwan e Department of Entomology, National Taiwan University, Taipei, Taiwan b c

a r t i c l e

i n f o

Article history: Received 18 July 2013 Revised 24 October 2013 Accepted 10 November 2013 Available online 26 November 2013 Keywords: Resistin Transcription factor FOXO Preadipocyte Adipocyte

a b s t r a c t Resistin is known as an adipocyte-specific hormone that can cause insulin resistance and decrease adipocyte differentiation. It can be regulated by transcriptional factors, but the possible role of forkhead transcription factor FOXO1 in regulating resistin gene expression is still unknown. Using 3T3 fibroblast and C3H10T1/2 and 3T3-L1 adipocytes, we found that transient overexpression of a non-phosphorylatable, constitutively active FOXO1, but not the wild type of FOXO1 or a DNA binding-deficient FOXO1, activated resistin promoter-directed luciferase expression. However, transient overexpression of a dominant-negative FOXO1 inactivated resistin promoter activity and reduced resistin mRNA expression. These observations indicate that the action of FOXO1 on resistin gene expression requires the activation of FOXO1 and that the effect of FOXO1 depends on the phosphorylation and dephosphorylation of FOXO1. The FOXO1 protein target sites on the resistin promoter were localized to the proximal 3545 to 787 bp of 50 -flanking region of the resistin promoter. A chromatin immunoprecipitation assay also showed that FOXO1 bound the resistin promoter at nucleotide regions of 1539 to 1366 bp and 1016 to 835 bp, but not at the regions of 795 to 632 bp. Results of this study suggest that FOXO1 transcription factor likely activates the expression of adipocyte resistin gene via direct association with the upstream resistin promoter. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction Resistin is a cysteine-rich hormone that was first isolated from adipose tissues and found to link obesity to type II diabetes in rodents (Steppan et al., 2001). In particular, an anti-resistin antibody improves insulin sensitivity in obese mice, while the administration of exogenous resistin into normal mice causes glucose intolerance and hyperinsulinemia (Steppan et al., 2001). In addition, transgenic mice overexpressing a dominant negative form of resistin showed improved insulin sensitivity and increased adipogenesis (Kim et al., 2004). However, the involvement of resistin in obesity and insulin resistance in humans is still controversial. q Part of this work was presented at the 14th International Congress of Endocrinology, March 26–30, 2010, Kyoto, Japan. ⇑ Corresponding author. Address: Department of Life Sciences, College of Science, National Central University, Jhongli City, Taoyuan 32001, Taiwan. Fax: +886 (3) 4228482. E-mail address: [email protected] (Y.-H. Kao). 1 These authors contributed equally to this work. 2 Co-corresponding author.

0016-6480/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygcen.2013.11.018

Some studies have shown that resistin mRNA expression in adipose tissues of obese humans is higher than that in normal subjects (Mcternan et al., 2002), and that a single nucleotide polymorphism in the resistin gene promoter is associated with obesity (Engert et al., 2002) and diabetes (Pizzuti et al., 2002). Others found no relationship between resistin gene expression and body weight or insulin resistance (Koerner et al., 2005). One possible explanation for these disparate findings is the presence of various isoforms (Arco et al., 2003; Nohira et al., 2004) or dimers (Patel et al., 2004) of resistin. This contention may also explain the functional diversity of resistin in different systems. For example, resistin regulates fasted blood glucose levels, lipid metabolism, catecholamine release, inflammation, hepatic insulin resistance, and proliferation and activation of endothelial cells and smooth muscle cells (Arco et al., 2003; Banerjee and Lazar, 2003; Banerjee et al., 2004; Calabro et al., 2004; Koerner et al., 2005; Moon et al., 2003; Muse et al., 2004; Ort et al., 2005). The mechanisms of actions of resistin can inhibit insulin signaling of 3T3-L1 adipocytes through the induction of the gene expression of suppressor of cytokine signaling 3 (Steppan et al., 2005).

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Despite the importance of resistin, relatively little is known about the control of gene expression of resistin by transcriptional factors (Banerjee and Lazar, 2003; Chen et al., 2006; Felipe et al., 2004; Hartman et al., 2002; Lee et al., 2008; Seo et al., 2003; Shojima et al., 2002; Song et al., 2002; Steppan et al., 2001). Expression of the adipocyte resistin gene was found to be positively regulated by C/EBPa, ADD1/SREBP1, and the estrogen receptor (Chen et al., 2006; Lee et al., 2008; Seo et al., 2003; Song et al., 2002) and negatively regulated by PPARc (Song et al., 2002; Steppan et al., 2001) and the retinoic acid receptor (Felipe et al., 2004), suggesting the possible involvement of transcription factors in regulating resistin gene promoter activity. However, the results of these studies did not demonstrate any effect of forkhead transcription factor FOXO on resistin gene promoter activity by adipocytes. In vivo, FOXO1 haploinsufficiency protects from diet-induced diabetes in mice and reduces adipose resistin mRNA levels (Nakae et al., 2003). In murine 3T3-F442A fat cells, FOXO1, FOXO3, FOXO4, and resistin genes express higher during differentiation of fat cells, but FOXO1 mRNA levels are relatively higher than those of FOXO3 and FOXO4 (Nakae et al., 2003). FOXO1, PI3K, or AKT/PKB proteins have been reported to be essential signal transducers of insulin or IGFs in regulating other genes in 3T3-L1 adipocytes (Nakae et al., 2000, 2003, 2008; Obsil and Obsilova, 2008). In particular, FOXO1 phosphorylated by AKT/PKB after insulin and IGF treatments undergoes nuclear exclusion and is thus inactivated (Nakae et al., 2000, 2003, 2008). Different studies showed that resistin expression was markedly suppressed by insulin and IGF treatments (Chen et al., 2005; Kawashima et al., 2003; Shojima et al., 2002) and by overexpression of the PI3K kinase p110a catalytic subunit or AKT in 3T3-L1 adipocytes (Shojima et al., 2002). Taken together, the results of these studies have created much speculation surrounding the possible role of FOXO1 protein in regulating resistin gene promoter activity. Further studies are required to determine whether FOXO1 is able to bind to the resistin gene promoter in fat cells and whether it can activate resistin promoter activity in preadipocytes and/or adipocytes. In this study, we used the resistin promoter-directed luciferase reporter gene expression system to examine the influence of FOXO1 transcription factor on resistin gene promoter activity. We investigated whether FOXO1 binds to the resistin gene promoter and which regions of the resistin gene promoter are possible target sites of the FOXO1 factor. 2. Materials and methods

10% CS, 100 U/ml penicillin, and 100 mg/ml streptomycin (GibcoBRL) in a humidified atmosphere of 95% air and 5% CO2 at 37 °C (Hung et al., 2005; Hsieh et al., 2010; Ku et al., 2009, 2012). Medium was replaced every 2 days. Because serum components contain factors that facilitate 3T3-L1 and C3H10T1/2 differentiation from preadipocytes to adipocytes when cells are confluent, these cells were subcultured before reaching confluency. Because the 3T3-L1 was a determined fat cell line subcloned from the 3T3 cells (Green and Kehinde, 1974), and because 3T3-L1 and C3H10T1/2 cell lines were both derived from murine embryonic cells and could be induced to differentiate by using the same adipogenic protocol described below (Reznikoff et al., 1973; Tang et al., 2004), all of these three cell lines were used for this study. This would also help provide more evidence to strengthen the effect of FOXO1 on resistin promoter activity, as well as might help understand whether the FOXO1 effect varies with cell types. To obtained C3H10T1/2 adipocytes and 3T3-L1 adipocytes, we followed the methods of Chang et al. (2012), Nakae et al. (2003) and Wang et al. (2009) with some modifications to differentiate preadipocytes into adipocytes. Briefly, 2-day post-confluent C3H10T1/2 and 3T3-L1 preadipocytes (3  106 cells in a 10-cm plate) were treated with DMEM containing a final concentration of 2 lg/ml insulin, 1 lM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 10% FBS for 48 h. The time when the cocktail was removed was set at Day 0 differentiation cells. The medium was then changed to DMEM containing 10% FBS and 1 lg/ml insulin for an additional 2 days. The medium was changed to DMEM containing 10% FBS for an additional 6–10 days. With this protocol, greater than 90% adipocyte differentiation was achieved, as indicated by phenotypical appearance and triglyceride accumulation (Chang et al., 2012, 2013). Differentiated adipocytes expressed more resistin, adiponectin, and aP2 mRNAs than either preadipocytes or differentiating preadipocytes. 2.3. Plasmid constructions According to the consensus DNA sequence TTGTTTAC of the FOXO1 protein binding element (Nakae et al., 2008) and the sequence of the proximal resistin promoter (Song et al., 2002), resistin promoter appeared to have multiple FOXO1 response elements in the proximal base pairs of 50 -flanking sequence (Fig. 1). To understand the regulation of mouse resistin gene expression by FOXO1 transcription factor, the 3.73 kb of mouse resistin gene promoter isolated from 3T3-L1 adipocytes was cloned into a

2.1. Chemical reagents All materials (e.g., insulin, dexamethasone, and 3-isobutyl1-methylxanthine) were purchased from Sigma (St. Louis, MO) unless otherwise stated. DMEM, penicillin–streptomycin, calf serum (CS), fetal bovine serum (FBS), trypsin, agarose, the 1-kb plus DNA ladder marker, and the protein marker were purchased from GibcoBRL (New York, NY). FOXO1 and GST antibodies were obtained from Cell Signaling Technology (Danvers, MA), while goat anti-rabbit IgG–HRP was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The 30 -RACE system, Trizol, and Taq polymerase were purchased from Invitrogen Life Science Technologies (Carlsbad, CA). BglII and MluI restriction enzymes were purchased from New England BioLabs, Inc. (Ipswich, MA). 2.2. Cell culture 3T3-L1 cells (American Type Culture Collection, Manassas, VA; ATCC-CL-173™), NIH3T3 cells (ATCC-CCL-1658), and C3H10T1/2 (ATCC-CCL-226) cells were grown in DMEM (pH 7.4) containing

Fig. 1. Schematic diagram showing the 3.7-kb mouse resistin gene promoter, in which the proximal 3545, 1125, 786, or 451 bp of 50 -flanking sequence plus 185 bp of the first exon was cloned into a luciferase reporter gene-constructed pGL3 vector with BglII and MluI insertions.

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luciferase reporter gene-constructed pGL3 vector (Promega) with BglII and MluI insertions. The resistin promoter contained the proximal 3545 bp of 50 -flanking region plus 185 bp of the first exon. To identify the response elements targeted by FOXO1, we completed a series of 50 -end deletion of resistin promoter mutants, including the proximal 1125, 786, or 451 bp of 50 -flanking sequence plus 185 bp of the first exon. Deletion clones of this promoter were prepared by PCR and then constructed in the pGL3 vector with BglII and MluI insertions for directed cloning. The primers used for amplication of each fragment aree shown in Table 1. The expression vectors for human FOXO1 (hFOXO1), FOXO1-AAA (hFOXO1AAA), and FOXO1-H215R (hFOXO1-H215R) (gifts from Dr. William Seller, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA) were constructed in either pMSCV-neoEB or pcDNA3-GFP plasmid. Expression vectors for mouse FOXO14256 (mFOXO14256) constructed in pCMV5 plasmid was purchased from Addgene Inc. (Cambridge, MA).

0.47 mM luciferin). Luminescence was detected by a Clarity 2 luminometer (BioTEK, Winooski, VT). To study the effect of FOXO1 overexpression on C3H10T1/2 and 3T3-L1 adipocytes, these cells were transfected by electroporation per Liao et al. (2006) with some modifications. Briefly, Day 4 differentiating 3T3-L1 adipocytes (107 cells) and Day 4 differentiating C3H10T1/2 adipocytes (107 cells) were trypsinized and transiently transfected by BTX Harvard Apparatus-ECM830 electroporation (Holliston, MA) at 200 V with 90 lg of plasmid DNA mixtures per cuvette. Mixtures of plasmid DNA contained 30 lg reporter vector, 30 lg expression vector, and 30 lg pRL-TK vector. After 20 ms of electroporation, the cells collected from one cuvette were replated on a 12-well dish in triplicate for an additional 48 h. After incubation, the cells were lysed and then analyzed for luciferase activity as indicated above.

2.4. Transient transfection and luciferase reporter assay

As previously described by Liao et al. (2006) with some modifications, we used the method of confocal laser scanning microscopy (Instrumentation Center of National Central University, Taipei, Taiwan) to check for nuclear localization of wild type and mutant forms of FOXO1 that were constructed into the pcDNA3-GFP vector and then expressed in 3T3, C3H10T1/2, and 3T3-L1 cells. Briefly, the cells were plated on glass chamber slides and grown as described above. NIH3T3 cells and C3H10T1/2 preadipocytes were lipofectamine-transfected with plasmid DNA mixtures (reporter vector and expression vector) for 1 h, while 3T3-L1 adipocytes and C3H10T1/2 adipocytes were transfected with plasmid DNA mixtures by electroporation as above. Cells were transfected with an empty pcDNA3-GFP expression vector as the control. After 48 h of cotransfection, cells were washed with 10 mM PBS and then fixed with 3% paraformaldehyde in PBS. Fixed cells were washed with PBS, stained by DAPI, and visualized with a confocal laser scanning microscope using a Leica TCS SP system with a 200 lens. Confocal images were scanned using the same settings for each experiment.

We carried out the transient transfections as previously described by Chang et al. (2006) and Hung et al. (2005). 3T3 and C3H10T1/2 preadipocytes (3  104 cells/well) were plated into 12-well culture dish with DMEM supplemented with 10% FBS. After allowing 12 h for attachment, the medium was then replaced with fresh DMEM containing mixtures of liposome (TransFastTM Transfection Reagent, Promega Corporation, Madison, WI) and plasmid DNA. Liposome was mixed with plasmid DNA in the HEPES buffer (pH 7.0) containing final concentrations of 20 mm HEPES, 137 mm NaCl, 5 mM KCl, 1 mm Na2HPO4, 5.5 mm dextrose, and the DNA mixtures (0.33 lg reporter vector, 0.33 lg FOXO1 expression vector, and 0.67 lg pRL-TK vector per well). After allowing 15 min for incubation at room temperature, the DNA/ liposome complex formed. After 1 h of cotransfection of the expression vector with the resistin promoter-directed luciferase reporter vector, the medium was then replaced with fresh DMEM containing 10% FBS for another 48 h. This allowed the FOXO1 protein to be expressed in cells. Cells were cotransfected with the Renilla luciferase control vector pRL-TK (Promega) to normalize results for transfection efficiency, or with an empty expression vector to correct total DNA amount. After incubation, cells were washed with 10 mM PBS and then lysed with a buffer (pH 8.0) containing 25 mM Tris–phosphate, 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N,N,N0 ,N0 -tetraacetic acid, 10% (v/v) glycerol, and 1% (v/v) Triton X-100. After centrifugation, 100-ll aliquots of total lysates were used to determine luciferase activity as an indication of luciferase expression, which was performed at 37 °C for 10 min in a final volume of 200 ll per well containing 100 ll of luciferin substrate solution (20 mM tricine, 1 mM magnesium carbonate hydroxide pentahydrate, 2.7 mM magnesium sulfate, 0.1 mM EDTA, 33 mM DTT, 0.27 mM coenzyme A, 0.5 mM ATP, and

2.5. Confocal laser scanning microscopy

2.6. Chromatin immunoprecipitation (ChIP) assay The ChIP method was adapted from Chen et al. (2006), Hartman et al. (2002), and Ku et al. (2009, 2012) with some modifications to analyze the association of mFoxO1 with the resistin gene promoter. Cells were collected by washing twice with PBS and crosslinking with 1% formaldehyde in PBS at 37 °C for 10 min. Cells were then rinsed twice with ice-cold PBS, centrifuged for 4 min at 1500 g, and resuspended in buffer I (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.1% NP-40, a tablet of protease inhibitor). Following 10 min of incubation on ice, samples were sonicated once at 90 s pulses on ice. Lysates were centrifuged at 600g for 10 min, and the collected nuclear pellet was then resus-

Table 1 Primers for resistin promoter-directed luciferase reporter gene construct. Promoters (size)

Sequence of primer

Accession No.

3545/+185 (3730 bp)

FP: 50 -CGACGCGTCGCTCTTGGAGCTGATGGAAGTTGA-30 RP: 50 -GAAGATCTTCGGGGAGTCAGACTGCAATGTCC-30

NC_000074

1125/+185 (1310 bp)

FP: 50 -CGACGCGTCGTTCTTGGCTACACAGTTCA-30 RP: 50 -GAAGATCTTCGGGGAGTCAGACTGCAATGTCC-30

NC_000074

786/+185 (971 bp)

FP: 50 -CGACGCGTCGGATAAGGCAGATTCTATTGAG-30 RP: 50 -GAAGATCTTCGGGGAGTCAGACTGCAATGTCC-30

NC_000074

451/+185 (636 bp)

FP: 50 -CGACGCGTCGAGACCATCTGTATAGAGCAT-30 RP: 50 -GAAGATCTTCGGGGAGTCAGACTGCAATGTCC-30

NC_000074

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pended in buffer II (1% SDS, 10 mM EDTA, 50 mM Tris–HCl, pH 8.1) with protease inhibitors (Roche Molecular Biochemicals). Samples were pre-cleared with 16 lg of sheared salmon sperm DNA and 80 ll of protein A-agarose beads (Santa Cruz Biotech) for 2 h. They were then immunoprecipitated with FOXO1 antibody (1 lg) or with normal rabbit immunoglobulin (IgG, 1 lg). After incubation overnight, samples were then incubated with 60 ll of protein A-agarose beads for 1 h followed by 4 min of sequential washes in buffer III (20 mM Tris–HCl, pH 8.1, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl), buffer IV (20 mM Tris–HCl, pH 8.1, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl), buffer V (10 mM Tris–HCl, pH 8.1, 0.25% M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA), and Tris–EDTA buffer (10 mM Tris–HCl, pH 8.0, 1 mM EDTA). Precipitates were extracted by incubating with an elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature for 15 min and then added to 20 ll of 5 M NaCl at 65 °C for 6 h. The extract was proteolysed by incubating with buffer VI (40 mM Tris–HCl, pH 6.5, 10 mM EDTA and 20 lg proteinase K) at 45 °C for 1 h. DNA fragments were purified using an Illustra GFX PCR DNA and Gel Band Purification kit (GE Healthcare Bio-Sciences Co., Piscataway, NJ). An aliquot of 5 ll of purified sample was used in either 35 cycles of RT-PCR or 40 cycles of real-time PCR. According to the method by Chen et al. (2006), the primers surrounding the resistin transcription start site had sequences as indicated in Table 2. 2.7. RNA analysis We used semi-quantitative real-time PCR to determine the mRNA levels of FOXO1, resistin, adiponectin, aP2, and perilipin (Chang et al., 2012, 2013). Total RNA was isolated with the Trizol kit, and cDNA was then synthesized from equal amounts (5 lg) of RNA using 100 units of M-MLV reverse transcriptase (Invitrogen) in the presence of 40 units of RNase inhibitor (Invitrogen). Real-time PCR analysis was performed twice in duplicate using power SYBR green PCR master mix and the ABI 7300 Sequence Detection System (Applied Biosystems, Foster City, CA) under the

following conditions: an initial denaturing cycle at 95 °C for 10 min, followed by 40 cycles of amplification consisting of denaturation at 95 °C for 3 s and annealing at 60 °C for 30 s. Forward and reverse primers are shown in Table 3. Normalization involved GAPDH mRNA levels as controls in parallel reactions. The relative expression ratio of FOXO1, resistin, adiponectin, and aP2 transcripts to GAPDH transcript were calculated (Chang et al., 2012, 2013) and then expressed as a percent of the control. To validate amplification specificity, a dissociation curve analysis was performed from 60 to 95 °C at the rate of 0.1 °C/s after the PCR and was indicated by a single peak of FOXO1, resistin, adiponectin, or aP2. The amplification efficiency for each FOXO1, resistin, adiponectin, and aP2 amplicon was close to or greater than 91%. In some experiments, FOXO1, resistin, adiponectin, and aP2 transcripts were semi-quantitatively visualized using a reverse transcription PCR (Chang et al., 2012; Chen et al., 2006). PCR was performed under the following conditions: an initial denaturing cycle at 95 °C for 5 min, followed by 25–30 cycles of amplification consisting of

Table 3 Primers for the detection of the binding of resistin promoter with FoxO1 transcription factor by ChIP assay. Promoters (size)

Sequence of primers

Accession No.

Resistin 1539/1366 bp FP: 50 -CCCTGGTTCCTCTCATTCTGA-30 RP: 50 -TGGCCATCGCCAACATAGTA-30 1016/835 bp FP: 50 -TATAAACTTCCTCTGCTAATCTCA-30 RP: 50 -ACGTGTGGGTGTAAGTGC-30 795/632 bp FP: 50 -GACAGAGAAGATAAGGCAGATTC-30 RP: 50 -TTTCTGCTTTCTGGTGCAA-30 Adiponectin 626/485 bp Perilipin 511/293 bp

FP: 50 -GTATGGGATCCGGTCTAGCA-30 RP: 50 -ATTCCCAGCACCCACAGTAA-30

NC_000074 NC_000074 NC_000074

NC_000082

FP: 50 -GGTAATTGATCTTGATAGCTATAGAC-30 NC_000073 RP: 50 -CACTGCAATGTGTGCCATTAAACTT-30

Table 2 Primers for the detection of FoxO1, resistin, adiponectin, and aP2 gene expression by RT-PCR and real-time PCR. Genes

Sequence of primer

Accession No. (size)

FP: 50 -ACGTGCATTCCCTGGTGTAT-30 RP: 50 -TCATTGTGGGGAGGAGAGTC-30

NM_019739 (399 bp)

Resistin

FP: 50 -GTACCCACGGGATGAAGAACC-30 RP: 50 -GCAGAGCCACAGGAGCAG-30

NM_022984 (252 bp)

Adiponectin

FP: 50 -AGAGAAGGGAGAGAAAGGAGATGC-30 RP: 50 -TGGTCGTAGGTGAAGAGAACGG-30

NM_009605 (379 bp)

aP2

FP: 50 -ACAAAATGTGTGATGCCTTTGTGGGAAC-30 RP: 50 -TCCGACTGACTATTGTAGTGTTTGATGCAA-30

NM_024406 (460 bp)

GAPDH

FP: 50 -CCTCTGGAAAGCTGTGGCGT-30 RP: 50 -TTGGCAGGTTTCTCCAGGCG-30

NM_008084 (189 bp)

FP: 50 -TCCCACACAGTGTCAAGACTACAA-30 RP: 50 -CTGCTGTCAGACAATCTGAAGGA-30

NM_019739 (97 bp)

Resistin

FP: 50 -AAGCCATCGACAAGAAGATCAAA-30 RP: 50 -TCCAGCAATTTAAGCCAATGTTC-30

NM_022984 (80 bp)

Adiponectin

FP: 50 -AAGGGCTCAGGATGCTACTGTT-30 RP: 50 -AGTAACGTCATCTTCGGCATGA-30

NM_009605 (76 bp)

aP2

FP: 50 -CAGAAGTGGGATGGAAAGTCG-30 RP: 50 -CGACTGACTATTGTAGTGTTTGA-30

NM_024406 (168 bp)

GAPDH

FP: 50 -GGAGCCAAAAGGGTCATCATCTC-30 RP: 50 -GAGGGGCCATCCACAGTCTTCT-30

NM_008084 (232 bp)

RT-PCR experiment mFOXO1

Real-time PCR experiment mFOXO1

C.-W. Liu et al. / General and Comparative Endocrinology 196 (2014) 41–51

denaturation at 95 °C for 1 min and annealing at 60–62 °C for 1 min, and extension at 72 °C for 1 min. A final extension cycle at 72 °C for 10 min was added after the last cycle. The PCR product was run on 2% (w/v) agarose gel electrophoresis using 40 mM Tris–acetate buffer (pH 8.0) containing 1 mM EDTA and visualized by 0.5 lg/ml ethidium bromide. The forward and reverse primers, as well as the cycles of amplification and the predicted sizes of PCR products, are listed in Table 3. An exponential range in the number of PCR amplifications for FOXO1 and resistin was observed between 25 and 40 cycles when compared to the GAPDH standard with an exponential range of 15–30 cycles. We used 20 cycles of amplification for GAPDH. The control for amplification of genomic DNA was that an equivalent amount of total RNA was not performed with a reaction of reverse transcription but amplified in the PCR reaction. Then, samples shown free of genomic contamination were further analyzed with those listed forward and reverse primers in Table 3 that were designed on the basis of different exons of FOXO1, resistin, adiponectin, aP2, and GAPDH, respectively.

2.8. EMSA We performed the EMSA as previously described by Chang et al. (2006). Briefly, double-stranded oligonucleotides corresponding to the resistin promoter region were prepared by PCR with KOD DNA polymerase (Toyobo Co., Ltd., Japan) and with forward and reverse primers shown in Table 4. These purified blunted DNAs were then labeled with polynucleotide kinase (PNK) in the presence of 50 lCi [c-32P]-ATP (specific activity of 6000 Ci/mM). The 32P-Labeled probe was purified by PAGE (15% gel) in a Tris–borate solution containing 45 mM Tris–borate and 1 mM EDTA. Bacterially expressed GST-FOXO1 proteins (5 lg) were used to bind to this [32P]-labelled oligonucleotide probe in a binding solution containing 25 mM HEPES (pH 7.4), 5 mM MgCl2, 4 mM EDTA, 2 mM DTT, 110 mM NaCl, 5 lg/ml BSA, 0.8% Ficoll, 1 lg poly(dIdC), and 0.1 lg salmon sperm DNA at room temperature for 30 min. Protein and DNA complexes were resolved by 5% PAGE in a 5% low ionic gel, containing 5% (v/v) glycerol, at 4 °C for at least 3.5 h. Signals on the gels were viewed by autoradiography.

Table 4 Primers for the detection of the binding of resistin promoter with hFOXO1 transcription factor by EMSA analysis. Promoter region

Sequence of primer

Accession No.

1485/1386

FP: 50 -GACACAAAACCAGGTGCTTT-30 RP: 50 -CACAGCTGCTTTGGGGGTCT-30 FP: 50 -GACACAAAACCAGGTGCTTT-30 RP: 50 -TGAGAAACTTCCTTTCAAAG-30 FP: 50 -CCTCAGGTCTCCAGCAATGC-30 RP: 50 -CACAGCTGCTTTGGGGGTCT-30 FP: 50 -TTGCATAGCTTACAAAAGAG-30 RP: 50 -CAGAGGAAGTTTATAAATTG-30 FP: 50 -TCTTTTCATTTGTCCAATTT-30 RP: 50 -TAATTAAAATAATAAATCTT-30 FP: 50 -TCTTTTCATTTGTCCAATTT-30 RP: 50 -TCCGGCTCTGAAGCTGTTAC-30 FP: 50 -TCTTTTCATTTGTCCAATTT-30 RP: 50 -TAATTAAAATAATAAATCTT-30 FP: 50 -ATAAACTTCCTCTGCTAATC-30 RP: 50 -GCAAATCCTGGGTGTTAACG-30 FP: 50 -ACAGCCTGACGTTAACACCC-30 RP: 50 -CGTGTGGGTGTAAGTGCGTG-30 FP: 50 -CAGCCTGACGTTAACACCCA-30 RP: 50 -TAATTAAAATAATAAATCTT-30 FP: 50 -TTGCCTAGACTCCTCCAGTG-30 RP: 50 -AGAAAGGACCCACTGAGAAA-30

NC_000074

1485/1436 1435/1386 1102/1002 1035/696 1035/936 1035/836 1015/908 936/836 935/696 594/525

NC_000074 NC_000074 NC_000074 NC_000074 NC_000074 NC_000074 NC_000074 NC_000074 NC_000074 NC_000074

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2.9. Statistical analysis Data are expressed as the mean ± SE. The unpaired Student’s t-test was used to examine differences between empty vector-transfected and FOXO1 expression vector-treated groups. One-way ANOVA followed by the Student–Newman–Keuls multiple-range test were used to examine differences among multiple groups. Differences were considered significant at p < 0.05. Statistics were performed using SigmaStat (Jandel Scientific, Palo Alto, CA).

3. Results and discussion 3.1. FOXO1 factor activated the resistin gene promoter The promoter activity and expression of the resistin gene are regulated by a variety of transcriptional factors and of coactivator systems (Banerjee et al., 2004; Chen et al., 2006; Felipe et al., 2004; Hartman et al., 2002; Lee et al., 2008; Seo et al., 2003; Shojima et al., 2002; Song et al., 2002; Steppan et al., 2005). To fully understand how FOXO1 regulates resistin gene expression, we initially studied whether FOXO1 affects promoter activity. We used the resistin promoter-constructed luciferase gene reporter pGL3 plasmid and co-transfected this plasmid with hFOXO1-AAA-encoding pMSCV vector in 3T3 fibroblasts (Fig. 2). We found that the expression of constitutively active hFOXO1-AAA increased resistin promoter-directed luciferase expression in 3T3 cells in a concentration-dependent manner (Fig. 2) when a pGL3-luciferase reporter system was constructed with the proximal 3545 bp of 50 -flanking region plus 185 base pairs of the resistin promoter (Fig. 2A). The activation concentration of hFOXO1-AAA required to increase luciferase expression was approximately 0.33–0.67 lg relative to the values of the promoter-transfected group without hFOXO1-AAA co-transfection. In addition, deletion of the 3545 to 1126 regions had no effect on the hFOXO1-AAA-stimulated activation of luciferase expression (Fig. 2B). However, deletions of the 3545 to 787 regions (Fig. 2C) and the 3545 to 452 regions (Fig. 2D) significantly blocked the hFOXO1-AAA-mediated activation of luciferase expression. As the functional domains of the FOXO1 protein were found to contain a consensus sequence for AKT/PKB phosphorylation sites (RXRXXS/T) and the phosphorylated FOXO1 protein undergoes nuclear export and is thus inactivated (Nakae et al., 2000, 2003; Obsil and Obsilova, 2008), we wanted to confirm that the hFOXO1-AAA protein is located in the nucleus to stimulate the resistin promoter. We transfected 3T3 cells with the wild type or the different mutant types of hFOXO1 plasmids constructed with the pcDNA3-GFP and then examined their location with the confocal microscope (Fig. 3A). In parallel, hFOXO1-H215R and hFOXO1-AAA-H215R mutants, which were mutated with the DNA binding deficiency, were also used. Indeed, we confirmed that hFOXO1 and hFOXO1-H215R proteins were located in the nucleus and cytoplasm, while FOXO1AAA and hFOXO1-AAA-H215R proteins were expressed in the nucleus but not in the cytoplasm. Next, we used the luciferase reporter gene pGL3 constructs containing the nucleotide regions of 3545 to +185 or 1125 to +185 of the resistin promoter in order to examine whether the wild type of hFOXO1 and other mutant hFOXO1 factors constructed with the pcDNA3-GFP regulate resistin promoter activity. At the region of 3545 to +185 bp of the resistin promoter, hFOXO1-AAA, but not the other hFOXO1 constructs such as hFOXO, hFOXO1-H215R, and hFOXO1-AAAH215R, significantly increased luciferase expression compared to the pcDNA control construct (Fig. 3B). At the region of 1125 to +185 bp of the resistin promoter, we found that the expression of hFOXO1, hFOXO1-H215R and hFOXO1-AAA-H215R mutants did

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Fig. 2. Exogenous cotransfection of human FOXO1-AAA (hFOXO1AAA) transcription factor and resistin promoter-constructed luciferase reporter plasmid to NIH3T3 cells stimulated luciferase activity when the resistin promoter was at regions between 3545 and 787 bp. Data are expressed as the means ± SE from triplicates of five experiments. Standard error bars are too small to be seen in A, B, and D. The control was represented without hFOXO1AAA treatment, which was constructed in the pMSCV vector. a–dGroups with different letters shown in A and B are significantly different (P < 0.05) from each other. In D, ⁄P < 0.05 vs. control.

not significantly alter luciferase expression; however, the hFOXO1AAA expression stimulated resistin promoter activation (Fig. 3C). To determine whether hFOXO1-stimulated resistin promoter activity also occurred in preadipocytes and adipocytes, we cotransfected C3H10T1/2 preadipocytes with the hFOXO1-encoding plasmid and resistin promoter-directed luciferase reporter construct and then measured the locations of different forms of hFOXO1, as well as changes in the luciferase activity (Fig. 4). Confocal analysis indicated that hFOXO1-AAA and hFOXO1AAA-H215R were located in the nucleus of both C3H10T1/2 preadipocytes (Fig. 4A) and that hFOXO1 and hFOXO1-H215R were present in the nucleus and cytoplasm. Similar locations of these wild-type and mutant FOXO1s were observed in C3H10T1/2 adipocytes (Fig. 4B) and NIH3T3 fibroblasts (Fig. 3A). In addition, we found that hFOXO1-AAA was more likely than hFOXO1, hFOXO1H215R, and hFOXO1-AAA-H215R to activate the resistin promoter at nucleotide regions 3545 to +185 (Fig. 4C) and 1125 to +185 (Fig. 4D). Similarly, there was greater activity in hFOXO1-AAA than in other forms of FOXO1 when C3H10T1/2 adipocytes were examined (Fig. 4E and F). Using 3T3-L1 adipocytes, we also observed that hFOXO1-AAA activated the resistin promoter at nucleotide regions 3545 to +185 and 1125 to +185 (Fig. 5A and B). In addition, none of the hFOXO1, hFOXO1-H215R, and hFOXO1-AAA-H215R had any significant effect on luciferase expression. These results are consistent with those observed in C3H10T1/2 adipocytes. Like C3H10T1/2 preadipocytes (Fig. 4A) and adipocytes (Fig. 4B), similar locations of these wild-type and mutant FOXO1s were observed in 3T3-L1 adipocytes (Fig. 5C). Together with these observations in 3T3 fibroblasts, C3H10T1/2 cells, and 3T3-L1 cells, FOXO1 was found to stimulate the resistin gene promoter. The overexpression of a nonphosphorylatable, constitutively active FOXO1AAA but not the wild type of FOXO1, is

effective in stimulating resistin promoter activity, suggesting the importance of the phosphorylation-and-dephosphorylation mechanism for the effect of FOXO1. 3.2. Dominant-negative mFOXO1 reduced resistin mRNA expression and promoter activity To demonstrate the role of FOXO1 in regulating resistin gene expression in fat cells, we transiently transfected C3H10T1/2 and 3T3-L1 adipocytes with pCMV5 expression vector encoding dominant-negative mFOXO1-D256 and then measured changes in resistin mRNA levels (Fig. 6). After we confirmed that mFOXO1 mRNA expression was increased following mFOXO1-D256 overexpression, we found that mFOXO1-D256 did reduce resistin mRNA levels in C3H10T1/2 (Fig. 6A) and 3T3-L1 (Fig. 6B) adipocytes in addition to suppressing adiponectin and aP2 mRNA expression. Using the resistin promoter-directed luciferase reporter gene expression in C3H10T1/2 and 3T3-L1 adipocytes, we further observed that mFOXO1-D256 significantly reduced luciferase gene expression in both types of cells when resistin gene promoter activity was examined at nucleotide regions of 3545 to +185 and 1125 to +185 (Fig. 6C and D). These results indicate that FOXO1 is required for base levels of resistin gene expression and promoter activity. 3.3. FOXO1 bound the resistin gene promoter Although FOXO1 is necessary for promoter activity and the expression of resistin in fat cells, the possibility that FOXO1 binds the resistin promoter remains to be established. To further demonstrate whether FOXO1 protein bound the resistin promoter (Fig. 7), we collected non-differentiating preadipocytes, day 2 differentiating preadipocytes, and differentiated adipocytes from C3H10T1/2

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Fig. 3. Transient overexpression of a non-phosphorylatable, constitutively active human FOXO1-AAA (hFOXO1AAA), but not the wild type of hFOXO1 or DNA binding-deficient FOXO1 mutants such as hFOXO1-H215R and hFOXO1AAAH215R, activated resistin promoter-directed luciferase expression in NIH3T3 cells when the resistin promoter was at regions between 3545 and 787 (B and C). (A) Confocal analysis indicated that hFOXO1AAA and hFOXO1AAAH215R were located in the nucleus of the NIH3T3 cells and that hFOXO1 and hFOXO1H215R were present in the nucleus and cytoplasm. (B and C) The proximal 3545 or 1125 bp of the 50 -flanking sequence plus 185 bp of the first exon was cloned into a luciferase reporter gene-constructed pGL3 vector when FOXO1 factors were expressively constructed in the pcDNA3.0-GFP (B and C) vector. Data are expressed as the means ± SE from triplicates of three experiments. Standard error bars are too small to be seen in (B and C). The control was represented by an empty pcDNA3.0 vector treatment in the presence of resistin promoter-luciferase reporter construct. In (B and C), ⁄P < 0.05 vs. control; §P < 0.05 vs. hFOXO1AAA.

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cells, and employed the ChIP assay method. In this assay, chromatin was isolated and subjected to cross-linking and shearing of the DNA prior to immunoprecipitation with specific antibodies against the mFOXO1 protein. The association of mFOXO1 protein with the resistin gene promoter was examined by RT-PCR (gel bands) and real-time PCR (bar graph) using primers specific for the resistin promoter (as described in Section 2) after the reversal of crosslinking. This analysis revealed that mFOXO1 of the non-differentiating C3H10T1/2 preadipocytes did not significantly bind to the resistin gene promoter, adiponectin gene promoter (the positive control; Qiao and Shao, 2006), or perilipin gene promoter (the negative control; Qiao and Shao, 2006). However, in differentiating preadipocytes and differentiated adipocytes, mFOXO1 significantly bound the resistin promoter at nucleotide regions of 1539 to 1366 bp and 1016 to 835 bp, but not at regions of 795 to 632 bp. Also, mFOXO1 did not bind to the 511 to 293 bp perilipin promoter, but did bind to the 626 to 485 bp adiponectin promoter. The observed association of mFOXO1 with the resistin gene promoter might explain the effect of FOXO1 on resistin promoter activity and gene expression. FOXO1 regulates the expression of adipocyte genes through direct and indirect pathways (Obsil and Obsilova, 2008). The direct pathway mediates FOXO1-stimulated expression of the adiponectin gene through a direct binding to the FOXO1-responsive element in this gene promoter (Qiao and Shao, 2006). The indirect pathway controls FOXO1-stimulated expression of the estrogen receptor by binding to other transcriptional factors (i.e., estrogen receptors), co-activators, and/or co-repressors (Obsil and Obsilova, 2008). In our study, FOXO1 appears to directly bind to the resistin promoter. This conclusion is based on the following observations. First, overexpression of the nucleus-localized form of and the DNA bindingdeficient mutant of hFOXO1-AAA-H215R did not alter resistin promoter activity, while overexpression of the nucleus-localized form of and the DNA binding-functional mutant of hFOXO1-AAA stimulated resistin promoter activity. Second, the ChIP assay indicated an association between mFOXO1 and the resistin gene promoter. Finally, when different fragments of the resistin promoter were end-labeled with 32P in a PNK-mediated reaction and the labeled products were then used as probes for detecting its binding by FOXO1 protein, our results obtained from an electrophoretic mobility shift assay (EMSA) indicated that the glutathione-S-transferase (GST)-hFOXO1 fusion protein expressed in bacteria bound to the nucleotide regions of 1485 to 1386, 1485 to 1436, 1435 to 1386, 1035 to 696, 1035 to 836, and 935 to 696, but not 594 to 525, of the resistin promoter (Fig. 8). These EMSA results are consistent with those observed FOXO1-binding regions of the resistin promoter from the ChIP data. It is unfortunate that the exact FOXO1-binding sequences in resistin promoter were not demonstrated in this study. Further studies are needed to verify FOXO1-binding sequences in resistin promoter using the methods of size-mutagenesis and DNA footprinting. We conclude that the transient overexpression of a nonphosphorylatable, constitutively active FOXO1 activated resistin promoter-directed luciferase expression in a dose-dependent manner. However, transient overexpression of either the wild type of FOXO1 or a DNA binding-deficient FOXO1 in the presence of serum increased luciferase expression much less or not at all. Dominantnegative mFOXO1-D256 inactivated resistin promoter activity and reduced resistin mRNA expression. FOXO1 bound the resistin promoter, and the FOXO1 protein target sites on resistin promoter were localized to the proximal 3545 to 787 bp of 50 -flanking region of the resistin promoter. Furthermore, EMSA indicated that GST-FOXO1 protein directly bound the nucleotide region of the resistin promoter at 1486 to 1387 and 935 to 696. These

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Fig. 4. Transient overexpression of a non-phosphorylatable, constitutively active human FOXO1-AAA (hFOXO1AAA), but not the wild type of hFOXO1 or DNA bindingdeficient FOXO1 mutants such as hFOXO1H215R and hFOXO1AAAH215R, activated resistin promoter-directed luciferase expression in C3H10T1/2 preadipocytes and adipocytes when the resistin promoter was at regions between 3545 and 787. (A and B) Confocal analysis indicated that hFOXO1AAA and hFOXO1AAAH215R were located in the nucleus of the C3H10T1/2 preadipocytes or adipocytes and that hFOXO1 and hFOXO1-H215R were present in the nucleus and cytoplasm. (C–F) The proximal 3545 or 1125 bp of 50 -flanking sequence plus 185 bp of the first exon was cloned into a luciferase reporter gene-constructed pGL3 vector when FOXO1 factors were expressively constructed in the pcDNA3.0-GFP vector. Data are expressed as the means ± SE. from triplicates of three experiments. Standard error bars are too small to be seen in (C–F). The control was represented by an empty pcDNA vector treatment in the presence of resistin promoter-luciferase reporter construct. ⁄P < 0.05 vs. control; §P < 0.05 vs. hFOXO1AAA. The size of the scale shown in (A and B) is 10 lm.

C.-W. Liu et al. / General and Comparative Endocrinology 196 (2014) 41–51

Fig. 5. Transient overexpression of a non-phosphorylatable, constitutively active human FOXO1-AAA (hFOXO1AAA), but not the wild type of hFOXO1 (hFOXO1) or DNA binding-deficient FOXO1 mutants such as hFOXO1H215R and hFOXO1AAAH215R, activated resistin promoter-directed luciferase expression in 3T3L1 adipocytes when the resistin promoter was at regions between 3545 and 787. (A and B) The proximal 3545 or 1125 bp of 50 -flanking sequence plus 185 bp of the first exon was cloned into a luciferase reporter gene-constructed pGL3 vector when FOXO1 factors were expressively constructed in pcDNA3.0-GFP vector. Data are expressed as the means ± SE from triplicates of three experiments, standard error bars are too small to be seen. The control was represented by an empty pcDNA vector treatment in the presence of resistin promoter-luciferase reporter construct. ⁄ P < 0.05 vs. control; §P < 0.05 vs. hFOXO1AAA. (C) Confocal analysis indicated that hFOXO1AAA and hFOXO1AAAH215R were located in the nucleus of the 3T3-L1 adipocytes and that hFOXO1 and hFOXO1-H215R were present in the nucleus and cytoplasm. The size of the scale is 10 lm.

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Fig. 6. Transient overexpression of the dominant-negative mouse FOXO1-D256 (m FOXO1D256) reduced resistin mRNA levels in C3H10T1/2 (A and C) and 3T3-L1 adipocytes (B and D) and suppressed resistin promoter-directed luciferase expression when the proximal 3545 or 1125 bp of 50 -flanking sequence plus 185 bp of the first exon was cloned into a luciferase reporter gene-constructed pGL3 vector. Data are expressed as the means ± SE from triplicates of three experiments. Standard error bars are too small to be seen. The control was represented by an empty pCMV5 vector and without mFOXO1D256 treatment. ⁄P < 0.05 vs. control.

data suggest that the FOXO1 transcription factor likely upregulates the expression of the adipocyte resistin gene via activation of the upstream resistin promoter. Because insulin and IGF were found to suppress resistin gene expression and inactivate FOXO1 activity in fat cells (Chen et al., 2005; Kawashima et al., 2003; Nakae et al., 2003; Shojima et al., 2002), results of this study showing the stimulatory effect of FOXO1 on resistin promoter activity from murine secondary predipocytes and adipocytes appear to explain the inhibitory effects of insulin and IGF.

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Fig. 7. Chromatin immunoprecipitation analysis indicated the association of FOXO1 transcription factor with resistin promoter in C3H10T1/2 and Day 2 differentiating adipocytes. Chromatin was isolated and subjected to cross-linking and DNA shearing prior to immunoprecipitation with either the specific antibody (Ab) against FOXO1 or the non-specific immunoglobulin (IgG). After immunoprecipitation, the levels of fragmented DNA were examined by RT-PCR (gel bands) and realtime PCR (bar graphs) using primers specific for the resistin promoter as described in Section 2. Bar graph data are expressed as the means ± SE from duplicates of three experiments after the analysis of real-time PCR, and are presented with the relative expression ratio of fragmented DNA in the FOXO1 Ab-immunoprecipitated group to that of fragmented DNA in the IgG-immunoprecipitated group. Control experiments were those subjected to IgG immunoprecipitation, while the levels of fragmented DNA not subjected to immunoprecipitation served as the input. ⁄ P < 0.05 FOXO1 Ab-immunoprecipitated group vs. IgG-immunoprecipitated group.

Fig. 8. FOXO1 directly binds to the resistin promoter. Bacteria expressed GSTFOXO1 (5 lg) was used to bind 32P-labelled DNA probes spanning the region 1485 to 525 in EMSA. The labeling of double-stranded oligonucleotides corresponding to the resistin promoter region were prepared by PCR with KOD DNA polymerase as described in Section 2 and with forward and reverse primers shown in Table 4. The arrowhead corresponds to the binding of FOXO1 to the promoter.

References Disclosure statement C.-W. Liu, S.-Y. Yang, C.-K. Lin, H.-S. Liu, L.-T. Ho, L.-Y.Wu, M.-J. Lee, H.-C. Ku, H.-H. Chang, R.-N. Huang, and Y.-H. Kao have nothing to declare. Acknowledgments We thank Drs. Shen-Liang Chen and Sheng-Ping Hsiao for their technical assistance, as well as Addgene Company for providing the mFOXO14256 vector. This work was supported by grants from the National Science Council, Taiwan (NSC 101-2311-B-008-002; NSC101-2918-I-008-004)); Taoyuan Armed Forces General Hospital (#9925 and #10221); National Central University and Cathay General Hospital Joint Research Foundation (102NCU-CGH-06); VGHUST Joint Research Program and Tsou’s Foundation, Taiwan (VGHUST99-P3-14); and National Central University and Landseed Hospital Joint Research Founction (NCU-LSH-102-A-001) to Y.-H.K.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ygcen. 2013.11.018.

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